Tissue sample preparation, and MALDI MS imaging thereof

ABSTRACT

Aspects of the present invention relate to a method for the preparation of samples for MALDI MS imaging. Certain embodiments relate to a method of matrix deposition for samples, wherein tissue sections are prepared via a synergistic combination of fixation with matrix. In certain embodiments, tissue is fixed with cold solvent, according to well-established histology protocols, and in the presence of matrix, allowing for high resolution spatial mapping of protein, lipid, sugar, and/or nucleic acid distribution. In certain embodiments, the present invention relates to fixation with matrix of whole organisms. In certain embodiments, animals are perfused with fixation and matrix mixtures, which allows for direct mass spectrometry analysis.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/302,126, now U.S. Pat. No. 8,945,941, which claims the benefit ofpriority to Patent Cooperation Treaty Application numberPCT/US2007/012597, filed May 24, 2007, which claims the benefit ofpriority to the filing date of U.S. Provisional Patent Application Ser.No. 60/808,755, filed May 26, 2006; the entirety of all of which arehereby incorporated by reference.

BACKGROUND OF THE INVENTION

Disease diagnosis and therefore treatment rely upon observations oftissue and cellular characteristics, such as proliferation, cellular andnuclear morphology, vascularization, and specific biomarkers. Forexample, both the choice of treatment and the risks taken duringsurgical resection of a tumor mass in the brain are dictated by tumortype and malignancy grade. Tangentially, detailed pathologycharacterization is revealing considerable overlap in the moleculesinvolved in one disease and the next, consistent with disease continuumrather than discrete disease states. For instance, the lines betweenAlzheimer's, Pick's, and Parkinson's diseases are often blurred (Galpernand Lang (2006) Ann Neurol 59:449), as are the lines between gradationsof a given tumor type (see Caprioli (2005) Cancer Res 65:10642; Schwartzet al. (2005) Cancer Res 65:7674; Iwadate et al. (2004) Cancer Res64:2496). As the list of overlapping molecules involved in cancers andother diseases grows, so does the need for comprehensive molecularprofiling as part of diagnosis (Schwartz, ibid; Schwartz et al. (2004)Clin Cancer Res 10:981). The recent adaptation of mass spectrometers andtheir respective computer applications to accommodate tissue analysis(Stoeckli et al. (2001) Nat Med 7:493) provides such a comprehensivemolecular profile (Schwartz, ibid; Iwadate, ibid; Rahman et al. (2005)Am J Respir Crit Care Med 172:1556; Yanagisawa et al. (2003) Lancet362:433; Chaurand et al. (2006) Curr Opin Biotechnol 17:431) and savestime compared to histology.

Mass spectrometry (MS) is a well-established technique used tocharacterize analytes by determining their molecular weight. Ordinarily,mass spectrometry involves the steps of: coating a sample presentationapparatus with an analyte, introducing the sample presentation apparatusinto the mass spectrometer, volatilizing and ionizing the molecules ofthe analyte, accelerating the ionized analyte toward a detector byexposing the ions to an electric and/or a magnetic field, and analyzingthe data to determine the mass-to-charge ratio of specific analyte ions.If an analyte remains intact throughout this process, data will beobtained that correspond to a molecular weight for the entire intactanalyte ion. Typically however, it is also desirable to obtain datacorresponding to the molecular weight of various fragments of theanalyte.

Matrix-Assisted Laser Desorption Ionization (MALDI) is an ionizationtechnique often used for mass spectrometric analysis of large and/orlabile biomolecules, such as nucleotidic and peptidic oligomers,polymers, and dendrimers, as well as for analysis of nonbiomolecularcompounds, such as fullerenes (Karas et al. (1987) Int. J. Mass.Spectrom. Ion Processes 78:53; Spengler and Kaufmann, (1992) Analusis20:91). MALDI is considered a “soft” ionizing technique, in which bothpositive and negative ions are produced. The technique usually involvesdepositing a small volume of sample fluid containing an analyte on asubstrate comprised of a photon-absorbing “matrix” material selected toenhance desorption performance. See Karas et al. (1988), “LaserDesorption Ionization of Proteins with Molecular Masses Exceeding 10,000Daltons,” Anal. Chem., 60:2299-2301. Said matrix material is usually acrystalline organic acid that absorbs electromagnetic radiation near thewavelength of the laser. When co-crystallized with analyte, the matrixmaterial assists in the ionization and desorption of analyte moieties.The sample fluid typically contains a solvent and the analyte. Once thesolvent has been evaporated from the substrate, the analyte remains onthe substrate at the location where the sample fluid has been deposited.Photons from a laser strike the substrate at the location of the analyteand, as a result, ions and neutral molecules are desorbed from thesubstrate. Prior to the development of MALDI, analysis of biomoleculesby mass spectrometry was quite difficult, if not impossible, since notechniques were available that were gentle enough to volatize intactbiomolecules without any degradation or fragmentation. MALDI techniquesare particularly useful in providing a means for efficiently analyzing alarge number of samples. In addition, MALDI is especially useful in theanalysis of minute amounts of sample that are provided over a small areaof a substrate surface.

Direct mass spectrometry analysis of a tissue sample affords a wealth ofchemical information, providing a molecular landscape of a tissue.Unlike current immunohistochemistry methods, which analyze theconcentration and distribution of only a single molecule per experiment,MS imaging provides information on hundred of molecules, affording abetter correlation between molecular composition and disease pathology,and therefore a more accurate diagnosis. Indeed, the ability to image asample with the objective to obtain the detailed spatial arrangement ofcompounds in an ordered target sample such as a slice of tissue usingMALDI MS would be of enormous value in biological research. For example,selected ion surface maps of such samples could provide details ofcompound compartmentalization, site-specific metabolic processing, andselective binding domains for a very wide variety of natural andsynthetic compounds.

Recently, mass spectrometry techniques involving laser desorption havebeen adapted for cellular analysis. For example, U.S. Pat. No. 5,808,300to Caprioli describes a method for imaging biological samples with MALDImass spectrometry. This method allows users to measure the distributionof a specific element or small molecule within biological specimens suchas tissue slices or individual cells. In particular, the method can beused for the specific analysis of peptides in whole cells, e.g., byobtaining signals for peptides and proteins directly from tissues andblots of tissues. In addition, the method has been used to desorbrelatively large proteins from tissues and blots of tissues in themolecular weight range beyond about 80 kiloDaltons. From such samples,hundreds of peptide and protein peaks can be recorded in the massspectrum produced from a single laser-ablated site on the sample. When alaser ablates the surface of the sample at multiple sites and the massspectrum from each site is saved separately, a data array is produced,which contains the relative intensity of any given mass at each site. Inthe MALDI MS imaging experiment (MSI), hundreds of closely spaced MALDIMS spectra are taken in a grid pattern where each spectrum is analogousto a pixel (Gusev et al. (1995) Anal. Chem. 67:4565; Stoeckli et al.(1999) J Am Soc Mass Spectrom 10:67; Caprioli et al. Anal. Chem. (1997)69:4751). Each pixel contains information on the mass and intensity ofhundreds of biomolecules, which can be translated into a spatial map ofmolecular distribution and abundance. An image of the sample surface canthen be constructed for any given molecular weight, effectivelyrepresenting a compositional map of the sample surface.

Accordingly, in order to perform mass spectrometry imaging, moleculesmust be transferred from the tissue and into the gas phase. As describedabove, this transfer requires a laser within the mass spectrometer, anda matrix which is applied to the tissue section. The matrix is a smallacid that crystallizes on the sample, and upon absorbing laser energy isvaporized along with molecules from the tissue. For efficient desorptionof large molecules (10 kDa and greater) by MALDI, sinapinic acid is acommonly used matrix. The matrix solution most often applied to tissuesamples is 20-30 mg sinapinic acid/ml and about 0.1% trifluoroaceticacid (TFA), in a solvent mixture of 50:50 volume ratio of acetonitrileto water. Hence, all current methods of matrix deposition on tissueslices, such as the solution base described, considerably perturb tissueintegrity, resulting in the solubilization and extraction of proteinsand peptides from the tissue and their subsequent diffusion frombiological location (Gusev, ibid.). Delocalization of proteins has adeleterious effect on image quality. Spatial resolution is lost, and theMS image becomes “blurred.” To minimize protein diffusion in the tissuesection, practitioners commonly use a method referred to as “spotting.”The goal of spotting is to apply very small droplets of solution, andsince proteins can only diffuse within the drop, the “blurring” islimited to smaller area. Spotting involves either manual depositionusing a pipette, or using a recently patented acoustic reagentmultispotter device (Aerni et al. (2006) Anal. Chem. 78(3):827-34; U.S.Pat. Nos. 6,707,038, 6,809,319, and 6,855,925 all to Ellson et al.). Thediameter of the spots is on average 1 mm, at least one thousand timestoo large for many histology-based diagnoses. Indeed, there areconsiderable drawbacks to this practice. Current methods of samplepreparation associated with this approach: (1) are expensive (the deviceof Caprioli, Ellson, et al. costs approximately $300,000, requiringsignificant capital investment); (2) are time consuming (hours vs.minutes to prepare and deposit sample); (3) do not provide images ofsufficient resolution (from 0.2 mm for the acoustic reagent multispotterto 1 millimeter resolution for manual deposition currently, whereas 1micrometer is needed); (4) do not provide a contiguous image or profileof the tissue; and (5) manual spotting is not reproducible.

Another approach used to cover tissue samples with matrix solutionwithout causing considerable protein displacement is referred to as“spraying.” This method consists of applying the matrix solutionuniformly on the tissue section using consecutively minimal volumes ofsolution to minimize the time that the tissue is in the presence of thesolvent. The spraying of matrix solution is typically achieved using anebulizer, paintbrush or modified electrospray source. Although somepractitioners precede spraying or spotting by ethanol fixation, theacetonitrile/water based solution of matrix can still dissolve anddisplace fixed proteins. Following our own attempts to improve sprayingusing a modified electrospray source (Schwartz et al. (2003) J MassSpectrom 38:699), we concluded that this method is still not suited foruse in a clinical setting. Although good images can be produced, imagequality was inconsistent and spatial resolution was operator-dependent.

Notwithstanding its promise, MS imaging has not yet been incorporated asa medical diagnostic, in part due to a lack of facile, reproduciblemeans of tissue preparation. A sample preparation method of matrixdeposition for tissue sections is needed for MALDI MS analysis, whichpreserves the spatial location of proteins during processing. Anobjective of the present invention and all attendant embodiments forMALDI MS imaging and sample preparation related thereto is to providemedicine with safe diagnostic strategies that are readily accessible toany established pathology or research laboratory.

SUMMARY OF THE INVENTION

Aspects of the present invention relate to a method for the preparationof samples for MALDI MS imaging. Certain embodiments relate to a methodof matrix deposition for samples, wherein tissue sections are preparedvia a synergistic combination of fixation with matrix. In certainembodiments, tissue is fixed with cold solvent, according towell-established histology protocols, and in the presence of matrix,allowing for high resolution spatial mapping of protein, lipid, sugar,and/or nucleic acid distribution. In certain embodiments, the presentinvention relates to fixation with matrix of whole organisms. In certainembodiments, animals are perfused with fixation and matrix mixtures,which allows for direct mass spectrometry analysis.

Aspects of the present invention provide the first such inexpensive,expeditious, and reproducible methodologies for sample preparation thatare compatible with histology protocols currently employed bypathologists for diagnosing diseases. Techniques provided by the presentinvention are broadly applicable toward the preparation of any samplefor MS imaging upon synergistic utilization of fixation and matrix andimprove the quality of spectra and provide images that do not have aperforated appearance, which marks an improvement over currentpractices. In certain embodiments, subcellular spatial resolution isachieved. The high-quality preparation and execution of methods of thepresent invention have the potential to improve diagnosis ofmalignancies involving a variety of biopsy types.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts confocal microscopy of different concentrations of matrixcrystals upon mouse brain tissue sections with a 20× dry objective,scale bar=75 μm: (a) 19.3 μm optical stack of a 25 mg/mL sinapinic aciddeposition by matrix solution fixation; (b-d) single optical slices (0.4μm) of three different matrix solution concentration depositions, 20-,25-, and 30 mg/mL sinapinic acid respectively.

FIG. 2 depicts a representative mass spectrum taken from a normal mousebrain tissue section of 16 μm thickness from 4000 to 20000 m/z preparedby sinapinic acid solution fixation. The spectrum was produced fromaccumulation of 500 laser shots and is not smoothed orbaseline-corrected. The inset magnifies the region of the spectrumbetween 7000 and 8200 m/z to represent data quality.

FIG. 3 depicts the immunohistochemistry of mouse brain tissue. Imagesare representations of optical stacks from the whole tissue thickness,unless otherwise specified. Cell nuclei are depicted using DAPI stainingand different proteins are depicted through labeling with an FITC orAlexa 488 secondary antibody probe, and visualized by confocalmicroscopy with a 63× oil objective; scale bars as indicated: (a-d)Comparison of the effect of different fixations on nuclei andneurofilaments integrity between standard paraformaldehyde (a), coldmethonal:ethanol:acetonitrile:about 0.1% TFA (b), cold acetone (c), andafter sinapinic acid solution fixation followed by matrix removal usingmethanol (d); (e-h) Distribution patters of a newly characterizedsoluble protein with distinctive tissue distribution, and perinuclearsubcellular localization. Tissue slices were prepared using cold acetone(e, f; 1-2 μm optical slices), standard paraformaldehyde fixation (g),and new cold methanol:ethanol:acentonitrile: about 0.1% TFA solution inaccordance with the present invention (h). Panels i and j compare thedistribution of MAP2 in mouse brain tissue fixed with paraformaldehyde(i) and with new matrix fixation solution at −20° C. (j).

FIG. 4 depicts electron microscopy of normal mouse brain cortical tissuesections at 6800× magnification: (a,b) Control sections that have notbeen treated prior to standard electron microscopy preparation. Cellularultrastructure preservation is observed; (c-f) Effect of the matrixsolution fixation process upon cellular ultrastructure. Panels c and dshow fixation with the cold ethanol:methanol:acetonitrile: about 0.1%TFA solution; panels e and f show fixation with cold acetone prior tostandard electron microscopy preparation. The treatments appear mostlyto affect cellular membranes to different degree. N indicates nuclei, Mis for membrane, and NM for nuclear membraine. Scale bars=2 μm.

FIG. 5 depicts (a,b) H&E staining of a pair of progressive meningiomasamples, grade II and III respectively, representing commonhistology-based tumor diagnosis. Light microscopy with 40× objective.The samples present heterogeneity that is observed in higher degree ofmalignancy, and the arrows are showing mitotic events. Samples werefixed with paraformaldehyde, paraffin embedded, and diagnosed by apathologist; (c,e,g) and (d,f,h) display MALDI MS molecular ion imagesof selected masses from a distinct pair of progressive meningiomasamples, grade II and III respectively. Scale bars=1 mm. Thedistribution of the ions presented also indicates heterogeneity, with aspecific molecular signature. Specimens were prepared by sinapinic acidsolution fixation (ethanol:methanol:acetonitrile:about 0.1% TFA) at −20°C. for 5 minutes.

FIG. 6 depicts confocal microscopy of neurofilament-FITC neurites fromsingle cells (bright threads) in mouse brain tissue prepared using coldacetone according to methods of the present invention: (A) 63×objective; (B) 20× objective.

FIG. 7 depicts (A) representations of the spatial distribution of threedistinct proteins of similar mass in a 100 μm resolution MALDI MS imageof a saggital mouse brain tissue slice prepared using ethanol/aceticacid according to methods of the present invention; (B) MS imagerepresentation overlaid upon.

DETAILED DESCRIPTION OF THE INVENTION

Aspects of the present invention relate to a method for the preparationof samples for analysis by MALDI MS imaging. This sample preparationmethod is compatible with the histology protocols currently employed bypathologists for diagnosing diseases.

Certain embodiments provide a protocol for matrix deposition of samples,e.g., tissue sections, which combines histology knowledge and massspectrometry analysis requirements, referred to herein as a “matrixsolution fixation” method, wherein fixation of said sample anddeposition of the matrix are combined in a single step. This methodprevents proteins from moving or diffusing from their original location;it has been successfully applied in major ways, and is being developedfurther to suit particular application requirements. In certainembodiments, the present invention relates to fixing tissue with coldsolvent, according to well-established histology protocols, and in thepresence of matrix. Said established immunohistochemistry fixationprotocols exploit specific chemical action such as protein denaturationat low pH, protein precipitation induced by solvents, and temperaturecontrolled protein diffusion. Aspects of the present invention alsoaddress the distinction between protein precipitation and agglomerationby providing for the optional selection of an appropriatesolvent/temperature combination, as agglomeration is more easilyreversible upon temperature increase.

In certain embodiments of the present invention, frozen tissue sections(e.g., brain and/or spinal cord) may be prepared and tested according tothe following solvent/matrix solutions: (a) acetone and 30 mg/mL ofeither sinapinic acid or α-cyano-4-hydroxycinnamic acid (HCCA) at −20°C.; (b) methanol and 30 mg/mL of either sinapinic acid or HCCA at −20°C.; (c) about about 95% ethanol : about 5% acetic acid combination and25 mg/mL of either sinapinic acid or HCCA at 4° C.; (d) about 50%methanol : about 50% acetone combination and 30 mg/mL of eithersinapinic acid or HCCA at −20° C.; (e) about 10% glacial acetic acid :about 30% chloroform : about 60% absolute ethanol combination and 30mg/mL of either sinapinic acid or HCCA; (f) about 50% methanol : about50% ethanol combination and 30 mg/mL of either sinapinic acid or HCCA at−20° C.; (g) 1 part methanol : 1 part ethanol : 1 part (about 50%acetonitrile : about 50% aqueous solution of about 0.1% trifluoroaceticacid) combination and 30 mg/mL of either sinapinic acid or HCCA at −20°C. and/or 4° C. In each case, the preceding histopathology fixationsolutions can be combined with matrix to provide MS imaging-readysamples. Optimum matrix crystal production and distribution and qualityof mass spectrometry data were observed using about 95% ethanol : about5% acetic acid combination and 25 mg/mL of either sinapinic acid or HCCAat 4° C. In certain embodiments, the present invention relates to amatrix solution fixation method for whole organisms. In animal studies,intact mice have been perfused with different “matrix solution fixation”mixtures, e.g., (a) Carnoy's fixative (10 mL glacial acetic acid : 30 mLchloroform : 60 mL absolute ethanol), and (b) about 95% ethanol : about5% acetic acid combination and 25 mg/mL sinapinic acid at 4° C.Perfusion of animals with fixative prior to mass spectrometry analysisdeprives the tissue of blood, and provides fixed tissue suitable fordirect mass spectrometry analysis. When animals are perfused with aCarnoy's/matrix solution, one is able to detect the presence of thematrix within the brain periphery by mass spectrometry. Thisdemonstration of tissue fixation and matrix incorporations using wholeanimal perfusion is remarkable.

Exemplary fixatives, commonly used in histology (i.e., histopathology,histochemistry, immunohistochemistry), include, but are not limited to:aldehydes (e.g., formaldehyde (paraformaldehyde, formalin),glutaraldehyde, acrolein (acrylic aldehyde), glyoxal (ethanedial,diformyl), malonaldehyde (malonic dialdehyde), diacetyl(2,3-butanedione), and polyaldehydes; alcohols (i.e., protein-denaturingagents; e.g., acetic acid, methanol, ethanol); heavy metal oxidizingagents (i.e., metallic ions and complexes; e.g., osmium tetroxide,chromic acid); agents of unknown mechanism, such as chloro-s-triazides,cyanuric chloride, carbodiimides, diisocyanates, diimido esters,diethylpyrocarbonate (diethyl oxydiformate, ethoxyformic anhydrate),picric acid, mercuric chloride (corrosive sublimate, bichloride ofmercury), and other salts of mercury, and acetone. What is more commonis to employ such agents in combination. Such combinations give rise tocommonly termed formulations known to those in the art, such as Carnoy'sfixatives, methacran, Wolman's solution, Rossman's fluid, Gendre'sfluid, Bouin's fluid, Zenker's fluid, Helly's fluid, B5 fixative, Susafluid, Elftman's fixative, Swank and Davenport's fixative, Lillie'salcoholic lead nitrate, and cetylpyridinium chloride (C.P.C.). Additivescan include, but are not limited to, such entities as tannic acid,phenol, transition metal salts (zinc), lanthanum, lithium, potassium.

MALDI MS matrix may be any material that absorbs light energy at afrequency easily accessible by a laser. It is further useful that saidmatrix materials gain physical access to biomolecules and are unreactivewith respect to biomolecules. Example matrices include, but are notlimited to: nicotinic acid, pyrozinoic acid, vanillic acid, succinicacid, caffeic acid, glycerol, urea or tris buffer (pH 7.3) and/orcombinations thereof. More commonly employed matrices includeα-cyano-4-hydroxycinnamic acid (HCCA), ferulic acid,2,5-dihydroxybenzoic acid, sinapinic (or sinapic) acid, 3,5-dimethoxy,4-hydroxy-trans-cinnamic acid, other cinnamic acid derivatives, gentisicacid, and/or combinations thereof.

Aspects of the present invention also relate to a method for samplepreparation using matrix-coated sample carriers, wherein a samplecarrier (e.g., a metal MALDI target, MALDI support, glass slide,ITO-coated glass slide, glass cover slip) is coated with matrix (i.e.,deposited), and sample tissue is the mounted upon said matrix-coatedcarrier and subsequently fixed with solvents (i.e., using the solutionsand methods of the present invention, or any solvent or solvent mixturethat will dissolve the deposited MALDI matrix). In certain embodiments,such a method involves the following steps: (1) coating any MALDI samplecarrier with matrix; (2) applying sample tissue to said matrix-coatedsample support; and (3) applying a solution that will fix the tissue(including but not limited to low temperature). In certain embodiments,it may be observed that deposition of the matrix material is easier andgives rise to better data and protects the instrument. Sample carriersdeposited with matrix material also facilitate the applications ofmethods of the present invention and the practical application ofembodiments of the present invention, including the packaging of suchmaterials in a diagnostic kit.

Selected Embodiments of the Invention

In certain embodiments, the present invention relates to a method forpreparing a sample for analysis by mass spectrometry, comprisingtreating, at a temperature, said sample with a solution comprising afixative and an analysis-enhancing material.

In certain embodiments, the present invention relates to theaforementioned method, wherein said analysis-enhancing material is firstdeposited onto a sample carrier to give a coated sample carrier.

In certain embodiments, the present invention relates to theabove-mentioned method, further comprising mounting said sample uponsaid coated sample carrier prior to treating with said fixative.

In certain embodiments, the present invention relates to the either ofthe above-mentioned methods, wherein said sample carrier is selectedfrom the group consisting of: metal targets, glass slides, or glasscover slips.

In certain embodiments, the present invention relates to theabove-mentioned method, wherein said sample carrier is a metal target;and said metal target is polished stainless steel or ground stainlesssteel.

In certain embodiments, the present invention relates to theabove-mentioned method, wherein said sample carrier is a glass slide orglass cover slip; and said glass slide or glass cover slip is coatedwith indium tin oxide (ITO).

In certain embodiments, the present invention relates to theaforementioned method, further comprising combining a fixative and ananalysis-enhancing material to give a solution.

In certain embodiments, the present invention relates to theaforementioned method, further comprising incubating said sample for aperiod of time, at a temperature, and drying said incubated sample.

In certain embodiments, the present invention relates to theaforementioned method, wherein the period of time is about 10 minutes.

In certain embodiments, the present invention relates to theaforementioned method, wherein the temperature is about −20° C.

In certain embodiments, the present invention relates to theaforementioned method, wherein the period of time is about 10 minutesand the temperature is about −20° C.

In certain embodiments, the present invention relates to theaforementioned method, further comprising rinsing said incubated sample,and drying said rinsed incubated sample, and optionally rinsing saidrinsed and dried incubated sample a second time, and drying saidoptionally rinsed sample.

In certain embodiments, the present invention relates to theaforementioned method, wherein the rinsing is done with a about 0.1%aqueous solution of trifluoroacetic acid (TFA).

In certain embodiments, the present invention relates to theaforementioned method, wherein said first rinse lasts a few seconds, andsaid second rinse lasts for about one minute.

In certain embodiments, the present invention relates to theaforementioned method, wherein the amount of about 0.1% aqueous solutionof TFA used is about 10-20 μL for each rinse.

In certain embodiments, the present invention relates to theaforementioned method, further comprising freezing said sample.

In certain embodiments, the present invention relates to theaforementioned method, wherein said fixative is a histology fixationsolution.

In certain embodiments, the present invention relates to theaforementioned method, wherein said fixative is selected from the groupconsisting of: (a) acetone; (b) methanol; (c) about 95% ethanol : about5% acetic acid combination; (d) about 50% methanol : about 50% acetonecombination; (e) about 10% glacial acetic acid : about 30% chloroform :about 60% absolute ethanol combination; (f) about 50% methanol : about50% ethanol combination; and (g) 1 part methanol:1 part ethanol:1 part(about 50% acetonitrile : about 50% aqueous solution of about 0.1% TFA)combination.

In certain embodiments, the present invention relates to theaforementioned method, wherein said fixative is about 95% ethanol :about 5% acetic acid combination.

In certain embodiments, the present invention relates to theaforementioned method, wherein the temperature ranges from about −100°C. to about 100° C.

In certain embodiments, the present invention relates to theaforementioned method, wherein the temperature ranges from about −78° C.to about 37° C.

In certain embodiments, the present invention relates to theaforementioned method, wherein the temperature is about −20° C., about4° C., about 37° C., or room temperature.

In certain embodiments, the present invention relates to theaforementioned method, wherein the temperature is about −20° C.

In certain embodiments, the present invention relates to theaforementioned method, wherein the temperature is about 4° C.

In certain embodiments, the present invention relates to theaforementioned method, wherein the temperature is about 37° C.

In certain embodiments, the present invention relates to theaforementioned method, wherein the temperature is room temperature.

In certain embodiments, the present invention relates to theaforementioned method, wherein said analysis-enhancing material is amass spectrometry matrix material.

In certain embodiments, the present invention relates to theaforementioned method, wherein said mass spectrometry matrix material isa photo absorbing matrix material.

In certain embodiments, the present invention relates to theaforementioned method, wherein said mass spectrometry matrix material isselected from the group consisting of: nicotinic acid; pyrozinoic acid;vanillic acid; succinic acid; caffeic acid; glycerol; urea buffer (pHabout 7.3); tris buffer (pH about 7.3); α-cyano-4-hydroxycinnamic acid(HCCA); ferulic acid; 2,5-dihydroxybenzoic acid; sinapinic acid;3,5-dimethoxy-4-hydroxy-trans-cinnamic acid; cinnamic acid derivatives;gentisic acid; and combinations thereof.

In certain embodiments, the present invention relates to theaforementioned method, wherein the analysis-enhancing material issinapinic acid or HCCA.

In certain embodiments, the present invention relates to theaforementioned method, wherein the analysis-enhancing material issinapinic acid.

In certain embodiments, the present invention relates to theaforementioned method, wherein the analysis-enhancing material is HCCA.

In certain embodiments, the present invention relates to theaforementioned method, wherein the analysis-enhancing material is at aconcentration of about 20-30 mg/mL.

In certain embodiments, the present invention relates to theaforementioned method, wherein the analysis-enhancing material issinapinic acid at a concentration of about 20-30 mg/mL.

In certain embodiments, the present invention relates to theaforementioned method, wherein the analysis-enhancing material is HCCAat a concentration of about 20-30 mg/mL.

In certain embodiments, the present invention relates to theaforementioned method, wherein the analysis-enhancing material comprisesa detectable label moiety.

In certain embodiments, the present invention relates to theaforementioned method, wherein said label moiety is fluorescent,radioactive, magnetic, biomolecular, or a combination thereof.

In certain embodiments, the present invention relates to theaforementioned method, wherein the sample is a whole organism.

In certain embodiments, the present invention relates to theaforementioned method, wherein the whole organism is an animal.

In certain embodiments, the present invention relates to theaforementioned method, wherein the animal is a mouse.

In certain embodiments, the present invention relates to theaforementioned method, wherein the sample is a cellular sample.

In certain embodiments, the present invention relates to theaforementioned method, wherein the sample is selected from the groupconsisting of a tissue sample, a cell culture, a single cell, cellularextract, or a plurality of cells immobilized on a substrate surface.

In certain embodiments, the present invention relates to theaforementioned method, wherein said tissue sample is animal tissue.

In certain embodiments, the present invention relates to theaforementioned method, wherein said animal tissue is mammalian tissue.

In certain embodiments, the present invention relates to theaforementioned method, wherein said mammalian tissue is primate, bovine,ovine, equine, porcine, rodent, feline, or canine.

In certain embodiments, the present invention relates to theaforementioned method, wherein the tissue is human.

In certain embodiments, the present invention relates to theaforementioned method, further comprising thaw-mounting said sample.

In certain embodiments, the present invention relates to theaforementioned method, wherein the thickness of the sample is less thanor equal to about 16 μm.

In certain embodiments, the present invention relates to theaforementioned method, wherein: the fixative is selected from the groupconsisting of: (a) acetone; (b) methanol; (c) about 95% ethanol : about5% acetic acid combination; (d) about 50% methanol : about 50% acetonecombination; (e) about 10% glacial acetic acid : about 30% chloroform :about 60% absolute ethanol combination; (f) about 50% methanol : about50% ethanol combination; and (g) 1 part methanol:1 part ethanol:1 part(about 50% acetonitrile : about 50% aqueous solution of about 0.1% TFA)combination; and the analysis-enhancing material is mass spectrometrymatrix material selected from the group consisting of: nicotinic acid;pyrozinoic acid; vanillic acid; succinic acid; caffeic acid; glycerol;urea buffer (pH about 7.3); tris buffer (pH about 7.3);α-cyano-4-hydroxycinnamic acid; ferulic acid; 2,5-dihydroxybenzoic acid;sinapinic acid; 3,5-dimethoxy, 4-hydroxy-trans-cinnamic acid; cinnamicacid derivatives; gentisic acid; and combinations thereof.

In certain embodiments, the present invention relates to theaforementioned method, wherein said fixative is about 95% ethanol :about 5% acetic acid combination, the analysis-enhancing material issinapinic acid or HCCA, and the temperature is about 4° C.

In certain embodiments, the present invention relates to theaforementioned method, wherein said fixative is about 95% ethanol :about 5% acetic acid combination, the analysis-enhancing material issinapinic acid, and the temperature is about 4° C.

In certain embodiments, the present invention relates to theaforementioned method, wherein said fixative is about 95% ethanol :about 5% acetic acid combination, the analysis-enhancing material isHCCA, and the temperature is about 4° C.

In certain embodiments, the present invention relates to a method ofmedical diagnosis comprising analyzing by mass spectrometry a sampleprepared according to any one of the methods described above.

In certain embodiments, the present invention relates to theaforementioned method, wherein the analysis is conducted by MALDI massspectrometry.

In certain embodiments, the present invention relates to theaforementioned method, wherein the analysis is conducted by MALDI MSimaging.

Aspects of the present invention are designed to prohibit proteins fromdiffusing during the process of MALDI matrix deposition, therebymaintaining their original location in sample tissue. As a measure ofsuccess, said tissue would maintain a heterogeneous distribution ofproteins, whereas current methods lead to homogeneity as previouslydescribed.

In certain embodiments, the present invention relates to the followinggeneralized methods and materials:

Tissue Slicing—In certain embodiments, sample tissues (e.g., mousebrains and/or spinal cords) are sectioned at 16 μm thickness,thaw-mounted on coverslips, and stored on dry ice until fixation. Saidsections are routinely processed from frozen tissue, yet may beharvested from tissue in any state.Preparation of Matrix Fixation Solution—In certain embodiments,solutions (supra) are prepared fresh prior to use. In certainembodiments, sinapinic acid (commercially available from Bruker orSigma) is dissolved in fixation solution (e.g., about 95% ethanol :about 5% acetic acid combination) by first vortexing for one minute,followed by sonication for 30 minutes. In certain embodiments, thesolution is then centrifuged for two minutes at maximum speed in amicrocentrifuge. In certain embodiments, the solution is then cooled to−20° C. and protected from light.Matrix Solution Fixation—In certain embodiments, tissue edges aredelineated with an ImmPenn. In certain embodiments, fixations describedherein are performed for 10 minutes at −20° C. In certain embodiments,the tissue slice is covered with the prepared cold matrix/fixativesolution (10-20 μL) and incubated for 10 minutes at −20° C. Said samplesare then optionally air-dried at ambient temperature. The tissue isrinsed with 10-20 μL of about 0.1% aqueous trifluoroacetic acid solutionfor a few seconds, then carefully removed with a pipette, and washed fora further one minute with 10-20 μL of about 0.1% aqueous TFA solution.The sample is then air-dried at ambient temperature.

The mechanical structures and/or equipment required for said analyticaltechniques (e.g., mass spectrometer) are well known to those skilled inthe art. Many aspects of the present invention are readily amenable toautomation and processing via high-throughput techniques, including, butnot limited to, parallel preparation and analysis (see, for example,U.S. Pat. No. 6,900,061). In certain embodiments, aspects of the presentinvention relate to the use of a sprayer, nebulizer, electrospraysource, or other source known to those in the art to deposit matrixmaterial while fixing the analyte tissue sample.

Fixation and Matrix Deposition

The greatest obstacle to preparing tissues for mass spectrometryanalysis by MALDI has been the conservation of tissue integrity andprotein localization upon matrix deposition. This is due to thesolubilization and displacement of proteins by the commonly used about50% acetonitrile (ACN): about 50% H₂O matrix solution. To overcome thislimitation, cold solvent tissue fixation protocols have been combinedwith matrix deposition. Starting from established immunohistochemistryprotocols, a series of fixative solutions have been developed thatincluded dissolved MALDI matrices, which have been used to fixconcomitantly the tissue and deposit matrix. This resulted in ahistology compatible method of tissue preparation for mass spectrometryimaging. The perturbation of tissue morphology, and protein distributionwere minimal, and optimized methods even maintained some subcellularstructure. Matrix crystals can be removed after imaging, providing theopportunity for histology and mass spectrometry imaging of the sametissue section. This method was tested and found to be compatible withcommon MALDI matrices, including HCCA, sinapinic acid, and2,5-dihydroxybenzoic acid (DHB). Frozen tissue sections were eitherthaw-mounted directly onto polished or ground stainless steel targets oronto glass- and indium tin oxide (ITO) coated glass coverslips. A numberof different solutions commonly used for histology fixation were foundto be useable for matrix solution fixation, including: (a) acetone; (b)methanol; (c) about 95% ethanol : about 5% acetic acid combination; (d)about 50% methanol : about 50% acetone combination; (e) about 10%glacial acetic acid : about 30% chloroform : about 60% absolute ethanolcombination (Carnoy's Fixative); and (f) about 50% methanol : about 50%ethanol combination. Each of the aforementioned solutions was evaluatedfor spectroscopic quality, reproducibility, effect on tissue morphology,and image quality. In general, solvent mixtures provided better spectrathan single solvent matrix solutions. Acetone combinations, forinstance, gave good spectroscopic data, but dehydrated and deformed thetissue on the micrometer scale. Washing or prefixing tissue with ethanolwas tested and found to be compatible with this matrix solutionfixation, although the ethanol wash resulted in a decrease in overallspectroscopic intensity (sum of signal/noise {S/N} for all peaks), andthe selective elimination of biomolecules.

Next, iterative optimization was undertaken of the following parameters:type and ratio of solvents; fixation time and temperature; dryingtemperature; matrix concentration; ratio of matrix solution to tissuevolume; and finally tissue thickness. Preparations that did notnoticeably perturb tissue morphology at the microscopic (10 micron ormore) level were then tested for spectroscopic quality. Spectroscopicquality was judged primarily as a function of the number of detectablepeaks, and then as a function of the signal to noise of individualpeaks, such that a spectrum with 10 peaks at S/N of 10 would beconsidered of higher quality than a spectrum with one peak at a signalto noise of 100. The following fixative solution was found to maximizeboth quantity and quality of analyte MS signals from tissue, whileminimizing diffusion of proteins and tissue deformation- ethanol:methanol: acetonitrile: about 0.1% TFA (in water) in a 2:2:1:1 ratio,containing 25-30 mg/ml of matrix. Fresh solution (less than 12 hoursold), and sonication of the matrix solutions for 30 minutes followed bya 2 minutes microcentrifugation at (15,000 g) were critical forreproducible and homogeneous crystallization (FIG. 1). Due to theirdelocalized π electron networks, matrix molecules have an inherent greenfluorescence, allowing their monitoring by fluorescence confocalmicroscopy. FIG. 1 illustrates matrix crystals upon a tissue section. Tomaintain a constant ratio of matrix solution to tissue volume, asurrounding physical and hydrophobic barrier approximately 3 mm awayfrom the tissue's edge was delineated using an ImmEdge Pen. With astandard fixation time of 5 minutes, followed by a room temperaturedrying time of approximately 5 minutes, the time required from slicingthe frozen tissue to introducing it to the mass spectrometer is 10minutes, producing high quality spectra, as illustrated in FIG. 2.

Spatial Resolution

To evaluate effects of optimized methods upon spatial resolution,treated normal mouse brain specimens were subject toimmunohistochemistry. FIG. 3 compares protein distribution in tissuesamples that were fixed with paraformaldehyde, acetone, or with theoptimized matrix solution (ethanol: methanol: acetonitrile: about 0.1%TFA in water in a 2:2:1:1 ratio). Confocal microscopy of nuclei usingthe fluorescent stain 4′,6-diamidino-2-phenylindole (DAPI), togetherwith antibodies against either neurofilament (FIG. 3a-d ), a newlycharacterized soluble protein (FIG. 3e-h ), or the soluble proteinmicrotubule associated protein 2 (MAP2) (FIG. 3i-j ) revealed intactgrey matter-white matter interfaces and axon bundles, and intact nucleiand neurofilaments. The sample shown in FIG. 3a was fixed withparaformaldehyde as a positive control for efficient tissue fixation,and presents a characteristic neuronal pattern. In comparison, samples 3b and c were respectively fixed with the optimized solution and acetone,both efficient solvents for matrix solution fixation, and show neuronalfeatures comparable to the paraformaldehyde control. FIG. 3d displays animage of a tissue section fixed with the optimized solution in thepresence of the matrix sinapinic acid. After crystallization, the matrixwas removed by solubilization in methanol. The neurofilament and nucleistaining indicates that matrix crystal formation as well as crystalremoval has minimal effects on tissue integrity. Samples in FIGS. 3e and3f were both fixed with acetone and stained for nuclei and a newlycharacterized soluble protein. Tissue integrity is observed by a cleardelineation between the two hemispheres and the upper commissure in FIG.3e and patterns of axon bundles in FIG. 3f . In FIGS. 3g and 3h , thesame antibody was used to illustrate preservation of a perinuclearlocalization by standard fixation with paraformaldehyde (FIG. 3g ) incomparison to fixation with our optimized solution (FIG. 3h ).Similarly, FIGS. 3i and 3j display preservation of the MAP2 proteindistribution upon treatment with either paraformaldehyde or an optimizedsolution of the present invention.

It was not possible to establish the matrix solution fixation imposedlimits of spatial resolution using confocal microscopy, although theresolution was judged to be less than 1 μm. To better estimatelimitations of spatial resolution that resulted from our treatment,electron microscopy, which exhibits at least 10 nm spatial resolution,was used. After a five minutes fixation at −20° C. of 16 μm sections ofnormal mouse brain tissue using the presentedmethanol:ethanol:acetonitrile: about 0.1% TFA solution (in the absenceof matrix) or acetone, the tissue was chemically fixed, embedded, andfurther sectioned for electron microscopy. Subcellular structures wereidentified, including nuclei, cytoplasmic ribosomes, rough endoplasmicreticulum, filaments, and some nuclear double membranes (FIG. 4).However, some cellular membranes were partially disrupted, thereforeminimizing delineation of subcellular compartments. Mitochondria, forexample, were less well defined in treated samples, indicating that ourmethod affects some sub-cellular structures. In regions allowingdistinction, cellular membranes appeared to dilate upon solventtreatment, going from approximately 40 nm thickness to roughly 90 nmthickness.

Imaging

Current tissue biopsy diagnostic procedures involve microscopic imagingto assess different tissue characteristics, such as cellularcomposition, morphology, proliferation, tissue vascularization, and uponavailability, the distribution of specific biomarkers. FIG. 5a and bshow the most common type of histological staining, a hematoxylin andeosin (H&E) staining of respectively grade II and III meningioma samplesresected from a patient with malignancy recurrence and progression afterinitial tumor removal. Hematoxylin stains nuclei dark blue or purplewhile eosin stains cytoplasm pink-orange, enabling cellular resolutionof some of the aforementioned histological characteristics. Somefeatures of grade II (FIG. 5a ) include prominent nucleoli, necrosis,and elevated mitotic rate (8 mitoses per 10 high power fields; a highpower field being the area of the specimen visible under highmagnification, 400× in this case), while the much higher mitotic rate(24-30 mitoses per 10 high power fields) observed in 5 b was enough toqualify as grade III. In comparison, 5 c, e, and g show selectedmolecular ions distribution over a grade II meningioma specimen, whiled, f, and h display the corresponding molecular ions over the grade IIImeningioma specimen from the same patient.

The MALDI MS images of FIG. 5 represents a given ion intensity at agiven location (for instance, the spatial distribution of a protein ofmass 15747 is shown in FIG. 5c and d ; a different ion mass is shown inFIG. 5e and f .) In this way, the molecular concentration as a functionof location is illustrated. The observed spatial resolution of the MSimages not only enables an assessment of tissue heterogeneity asobserved from the H&E staining but also provides a direct assessment ofa multitude of molecular features for comprehensive molecular diagnosis.

FIG. 6 depicts the results from an exemplification of certainembodiments of the present invention, wherein protein distribution ofneurofilament-FITC, a neuron-specific protein, has been accessedfollowing preparation of mouse brain tissue in cold acetone according tocertain embodiments the matrix solution fixation methods of the presentinvention detailed herein. Intact neurites of single-cells are clearlyvisible by confocal microscopy in FIG. 6A (bright, threadlikeappearance), indicating that fixation methods of the present inventionare not disruptive, effectively leaving cells whole (i.e., undamagedand/or unbroken). A broader field image (FIG. 6B) illustrates that themacroscopic properties of the tissue have also not been affected.

FIG. 7 illustrates that protein localization is not affected by matrixsolution fixation methods of the present invention, as distinctlocalization is maintained for three proteins of similar mass in a 100μm resolution MALDI MS image of a saggital mouse brain tissue sliceprepared using ethanol/acetic acid according to certain embodiments thematrix solution fixation methods of the present invention detailedherein. During MALDI MS analysis, the laser is rastered 100×100 μm overthe tissue slice, generating over 5000 position of analysis. Eachposition is then characterized by a mass spectrum of a selected massrange of interest. FIG. 7A depicts a representation of said spatialdistribution, and FIG. 7B further depicts a MS image representationoverlaid upon.

These experiments demonstrate the successful application of certainembodiments of the present invention, as the proteins are clearly notdiffuse (i.e., they are not evenly distributed across the entiretissue). Indeed, results obtained using the novelfixation/matrix-deposition methodology of the present inventionroutinely outrank all published data, extending the scope of thisdiscovery from a diagnostic method to a technique that may be practicedin all MS imaging applications and is applicable to the thousands ofacademic and industry practitioners of MALDI MS. The techniques providedby embodiments of the present invention improve upon the quality of theaverage spectra, and provide an image that does not have a perforatedappearance (i.e., the image is actually an agglomerate of many tinycircles that each resulted from deposition of one droplet), as do allimages that derive from spotting. Moreover, the approaches provided byembodiments of the present invention are inexpensive, very fast, andeasily reproducible. For the sake of comparison, the recently patenteddeposition device of Caprioli (supra) takes hours to deposit sample, andcosts approximately $300,000. Aspects of the present invention providemethods that require only minutes, and no additional equipment. Incertain embodiments, the methods of the present invention for tissuepreparation for MALDI mass spectrometry imaging make this type ofanalysis accessible to any established pathology or research laboratory.

The ability to image a sample and obtain the detailed spatialarrangement of compounds in an ordered target such as a slice of tissuewith MALDI MSI would be of enormous value in basic biological researchas well as clinical diagnosis. For example, selected ion surface mapscould provide details of compound compartmentalization, site-specificmetabolic processing and selective binding domains for a very widevariety of natural and synthetic compounds. To rank a new technology inthe imaging category, prepared samples must be a reasonable and ideallyan exact representation of the in vivo tissue. This concept wasrecognized over a hundred years ago and led to the development of amultitude of well-characterized histology methods for preparing tissuesfor microscopic study (see Clarke (1851) Phil. Trans. Roy. Soc. 141:601;Carnoy (1887) Cellule 3:6; Blum (1893) Vorlaufige Mittheilung. Z. w. M.10:314). Merging knowledge and understanding from this traditional fieldwith mass spectrometry has led us to develop the sample preparationmethods of the present invention in order to enable the routine,accurate, and comprehensive study of frozen tissue samples by MSI.Moreover, the newly developed matrix solution fixation methods providetissue with a level of integrity that meets standard histology-pathologyrequirements as well as an ease of execution that makes it possible toimplement in clinical settings. As the desorption and ionizationprocesses of MALDI mass spectrometry applications involve an energytransfer from a laser source to the analyte via a crystallized compoundreferred to as matrix, the latter must be incorporated onto the tissuesample of interest. One of the limitations preventing the broaderapplication of MSI has been the incompatibility of MALDI samplepreparations with histology requirements. This is because solventsystems and conditions routinely used to solubilize matrix compounds cansolubilize biomolecules and disturb histological and cellularstructures. Methods of the present invention preserve tissue and proteinstructure and enable the preparation of images for publication inaccordance with scientific guidelines for bioimaging applications(Editorial (2006) Nature Methods 3:237; Couzin (2006) Science 314:1866).

The matrix solution fixation (MSF) methods of the present invention arebased upon histology protocols known to prevent proteins from moving ordiffusing from their original location. It concomitantly denatures andprecipitates analytes, while incorporating the matrix onto the tissuefor further MSI analysis. Established histology fixation protocolsexploit specific chemical action such as protein denaturation at low pH,protein precipitation induced by solvents, and limited protein diffusionat low temperatures (Prausnitz (2003) Pure Appl. Chem. 75:859).

Decreasing the fixation temperature is expected to have the positiveeffect of minimizing diffusion during fixation, and the potentiallynegative effect of promoting agglomeration (easily reversibleprecipitation where protein denaturation does not occur). In contrast,fixation of smaller molecules such as peptides (<1500 amu) required theuse of acetone as matrix solvent, and the fixation had to be performedon dry ice to prevent diffusion. Independent of the deposition methodused (spraying, spotting, and matrix solution fixation), sonication andclarification by centrifugation of the matrix solution consistentlyrendered significantly higher quality spectra. Such a process produces amore homogenous solution and eliminates nucleation particles that areresponsible for inconsistencies in MALDI. Similarly, optimization of thematrix solution concentration also improved the deposition quality.

The methods of the present invention also afford flexibility inoptimizing crystallization parameters, such as crystal size, bymodulating the rate of solvent evaporation time, fixation/depositiontime, and total amount of matrix applied. A variety of conditions can beexploited to favor desorption and ionization of different classes ofbiomolecules based on their respective physico-chemical properties,enabling the investigation of other important biomolecules such as DNA,RNA, saccharides, and lipids. Application-specific design of fixationapproaches already enables a variety of molecular profiling studies(Gillespie et al. (2002) Am. J. Pathol. 160:449; Ahram (2003) Proteomics3:413). Solvent fixation treatment of frozen tissue sections is known tosolubilize lipids and cellular membrane structures, generally excludingthis treatment option for electron microscopy sample preparationmethods.

The sub-cellular disruption caused by our technique was ˜50-300 nm, farbelow the 10-150 μm resolution offered on commercial MALDI massspectrometers and on the order of the theoretical diffraction limit(λ/2) of the nitrogen laser used in this study. For direct massspectrometry analysis of complex tissue, the quality of rendered spectrareflects a balance between resolution and the quantity of informationextracted. The uniformity of tissue preparation following techniquesprovided by the present invention allows for direct analysis of anypoint of interest without subsequent data extrapolation. The MSF methodsof the present invention were also extended to whole animal bodyperfusion, with and without matrix, and are a convenient means ofpreparing tissue for MSI; it will also benefit animal model studies.

Methods of the present invention compare favorably with currently usedmatrix deposition in discrete droplet techniques such as acousticdeposition and noncontact microdispensors (Sloane et al. (2002) Mol CellProteomics 1:490). They exhibit similar spectroscopic quality andspatial resolution as well as a faster execution, improved from the220-300 μm range reported for acoustic reagent multispotter (ARM)process. In current practice however, the inherent resolution of saidtechniques cannot be realized in MS images due to a combination of massspectrometer sensitivity and laser focus. This limits the currentachievable resolution to around 10 μm, or a 20-fold improvement withhighly focused lasers. This opens a world of possibilities, includingimaging single cells like motor neurons; they have large cell bodydiameters, on the order of 40-100 μm. Moreover, it will enable higherresolution tissue imaging from ion microscope mode instruments, forwhich lateral resolutions ranging from 0.6-4 μm have been reported(Spengler and Hubert (2002) J. Am. Soc. Mass Spectrom 13:735; Luxembourget al. (2004) Anal. Chem. 76:5339; Luxembourg et al. (2005) J. ProteomeRes 4:671).

Methods of the present invention also compare favorably with animmersion method of matrix deposition that follows a tissue-washing stepwith organic solvents; in this method, the tissue is deprived of itslipids prior to standard matrix application (Lemaire et al. (2006) Anal.Chem. 78:7145). MSF preserves a higher level of tissue integrity throughmaintenance of the cellular ultrastructure and the general ensemble ofbiomolecules, including lipids. The new MSF methods of the presentinvention can also be extended to selectively favor distinct classes ofbiomolecules by taking advantage of intrinsic properties of distinctfixative solvents suitable for matrix dissolution. MSF methods combinehigh tissue integrity through temperature controlled diffusion kineticswith simultaneous crystallization of matrix and analytes duringextraction.

Methods of the present invention also compare well with the recentlydeveloped tissue preparation of adhering tissue upon beads embedded inparaffin, and then stretching the paraffin to the tissue over a largerarea (Monroe et al. (2006) Anal. Chem. 78:6826); both techniques arecurrently capable of the analysis of single, large cells. One advantageof the stretching technique is the large number of chemistries madeeasily available by changing the composition of the imbedded particle.Of all the techniques of MSI sample preparation, the embodiments of thepresent invention and the stretching technique are the only that couldconceivably be used to prepare samples for both MSI and for secondaryion mass spectrometry imaging (Lechene et al. (2006) J. Biol. 5:20;Monroe et al. (2005) JACS 127:12152), which has a resolution of around40 nm.

EXEMPLIFICATION

The invention may be understood with reference to the followingexamples, which are presented for illustrative purposes only and whichare non-limiting.

Materials

Swiss nude male mice of 4-8 weeks of age from Charles River Laboratories(Wilmington, Mass., USA) were anesthesized and sacrificed byintra-peritonal 90 mg/kg ketamine and 10 mg/kg xylazine. All animalmanipulations were carried out in the animal facility at the Brigham andWomen's Hospital in accordance with federal, local, and institutionalguidelines. Tumor samples were obtained from the Brain Tumor Bank in theDepartment of Neurosurgery, Brigham and Women's Hospital and analyzedunder appropriate Institutional review board guidelines. Calibrationstandards and α-cyano-4-hydroxycinnamic acid (HCCA) matrix were purchasefrom Bruker Daltonics (Billerica, Mass., USA). Sinapinic acid waspurchased from Sigma. The pan neuronal neurofilament monoclonal antibodywas from Abcam (Cambridge, Mass., USA), MAP2 polyclonal antibody fromChemicon (Billerica, Mass., USA), fluorescein (FITC)-conjugated donkeyanti-mouse secondary antibody was from Jackson ImmunoResearch (WestGrove, Pa., USA), and Alexa 488-conjugated donkey anti-rabbit secondaryantibody was from Invitrogen (Carlsbad, Calif., USA). The antibodyagainst the newly characterized protein was raised in rabbit (Agar etal, manuscript in preparation). The nucleic acid stain DAPI was fromCalbiochem (San Diego, Calif., USA). Chemicals (solvents are HPLC grade,acids and detergents) were from Fisher Scientific (Fair Lawn, N.J.,USA). Glass coverslips are 22×22 mm (thickness 1: 0.13-0.17 mm) fromFisher Scientific (Pittsburgh, Pa., USA). ITO coated coverslips withbusbars were 18×18 mm with resistivity from 8-12 ohms (thickness 1:0.13-0.17 mm) from SPI Supplies (West Chester, Pa., USA). ImmEdge PENfrom Vector Laboratories, Inc. (Burlingame, Calif., USA).

Sample Preparation for MALDI Mass Spectrometry Imaging

Mouse brain tissue and human tumor specimens were sectioned using aMicrom HM525 cryostat from Mikron Instruments Inc. (San Marcos, Calif.,USA). Method Optimization: In selecting a proper solvent combination fora given experiment, attention was also given to the surface tensioneffect on matrix distribution. For a specimen of dimensions 16 μmthickness with a surface of 10×5 mm, the tissue volume is 0.8 μl. Theratio recommended by the Online Information Center forImmunohistochemistry IHC World being of at least 20:1, we found thatusing a 20 μl volume of fixative/matrix solution was optimal. We notedthat the appearance of the matrix layer is a reliable indicator of itsionization/desorption efficiency potential. The most efficient matrixlayers had a homogenous and shiny appearance whereas scattered layerswith a mat finish rendered lower quality spectroscopic data. In ourunderstanding, the ability of the matrix layer to reflect light isindicative of the matrix crystals homogeneity.

Mass Spectrometry

A MALDI-TOF mass spectrometer Microflex interfaced with FlexImagingSoftware (inHouse version) from Bruker Daltonics (Billerica, Mass., USA)was operated in linear mode for m/z greater than 3000, and in reflectronmode for m/z less than 3000. Standard instrument parameter were used,although delayed extraction times were optimized to between 400-800 ns,depending upon the preferred mass range. To perform imaging of tissuespecimens directly from coverslips in a Microflex instrument withoutintroducing modifications of either the laser beam or molecular ionspath distances, we custom made coverslip holders from original Bruker'sMicroflex stainless steel target with a cavity of 25×25 mm with a depthof 300 μm was produced to accommodate glass coverslips of dimensions:22×22 mm, thickness 1 (0.13 to 0.17 mm). The coverslips are held on thetarget by minimal conductive adhesive tape.

Immunohistochemistry

Protocol to reduce background staining for mouse produced antibody onmouse tissue staining all performed at room temperature; tissue sectionsof 9 μm thickness. Blocking for 30 minutes with PBS solution: 2% normaldonkey serum, 1% BSA, about 0.1% gelatin, about 0.1% Triton X-100, 0.05%Tween-20, 0.05% sodium azide. Primay for 60 minutes in PBS solution: 1%BSA, 0.01% gelatin, 0.05% sodium azide, anti-NF 1:1000, anti-MAP21:1000, anti-new-protein 1:1000 (Agar et al., in preparation). Rinse 3times for 5 minutes with PBS. Secondary antibody for 45 minutes in PBSsolution 2% normal donkey serum, anti-mouse fluorescein (FITC)conjugated secondary antibody 1:200 and 4,6-diamidino-2-phenylindole(DAPI) 1 μg/ml. Wash twice with PBS for 10 minutes.

Confocal Microscopy

Immunohistochemistry was visualized on a Leica TCS SP2 AOBS (AccoustoOptical Beam Splitter) Spectral Confocal Microscope equipped with a 405nm UV laser. The UV laser was used to excite DAPI at 405 nm, and the 488nm line of the Argon laser was used to excite the FITC fluorophore.Samples were scanned at 0.275-0.623 micrometer optical slices (in the zdirection). Stack images of 10-16 slices were combined using the 2Dmaximum projection of a series along fixed axis option. Intensity andcontrast of each acquisition channel were readjusted for each sampleusing the “glow over under” option of the Leica Confocal Software.

Electron Microscopy

Samples were fixed in a mixture of 2.5% Glutaraldehyde and 2%Paraformaldehyde in 0.1 M sodium cacodylate buffer (pH 7.4), washed in0.1 M cacodylate buffer and postfixed with a mixture of 1%Osmiumtetroxide +1.5%

Potassium ferrocyanide for 30 minutes (control was 2 hours), washed inwater and stained in 1% aqueous uranyl acetate for 30 minutes (controlwas 1 hour) followed by dehydration in grades of alcohol (about 50%,70%, about 95%, 2×100%) and then infiltrated and embedded in TAAB Epon.(Marivac Canada Inc. St. Laurent, Canada)

Ultrathin sections (about 60-80 nm) were cut on a Reichert Ultracut-Smicrotome, picked up on to coppergrids, stained with 0.2% lead citrateand examined in a “Tecnai G² Spirit BioTWIN” Transmission electronmicroscope. Images were taken with a 2 k AMT CCD camera.

INCORPORATION BY REFERENCE

All of the U.S. patents and U.S. patent application publications citedherein are hereby incorporated by reference.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

We claim:
 1. A method of generating an image of a tissue sample, whereinthe image details the spatial distribution and abundance of compounds inthe tissue sample, comprising (i) combining a fixative with a massspectrometry matrix material, wherein the fixative is an aldehyde, analcohol, chloroform, acetic acid, trichloroacetic acid, acetone, water,cyanuric chloride, a carbodiimide, a diisocyanate, a diimido ester,2,3-butanedione, diethylpyrocarbonate, picric acid, tannic acid, phenol,cetylpyridinium chloride, a heavy metal oxidizing agent, FeCl3, amercury salt, lead nitrate, a zinc salt, lanthanum, lithium, potassium,potassium dichromate, potassium chlorate, sodium sulfate, sodiumacetate, or sodium chloride, or a combination thereof, and wherein themass spectrometry matrix material is nicotinic acid, pyrozinoic acid,vanillic acid, succinic acid, caffeic acid, glycerol, urea buffer, trisbuffer, a-cyano-4-hydroxycinnamic acid, ferulic acid,2,5-dihydroxybenzoic acid, sinapinic acid,3,5-dimethoxy-4-hydroxy-trans-cinnamic acid, cinnamic acid derivatives,or gentisic acid, or a combination thereof, thereby forming a fixativemixture; (ii) treating the tissue sample with the fixative mixture, at atemperature and for a period of time sufficient to inhibit compounds inthe sample from moving or diffusing from their original locationtherein, thereby forming a fixed sample; and (iii) imaging the fixedsample using MALDI mass spectrometry, thereby generating the image ofthe tissue sample.
 2. The method of claim 1, further comprisingincubating the tissue sample for a period of time, at a temperature, anddrying the incubated sample.
 3. The method of claim 1, furthercomprising freezing the tissue sample.
 4. The method of claim 1, whereinthe fixative is selected from the group consisting of: (a) acetone; (b)methanol; (c) about 95% ethanol:about 5% acetic acid combination; (d)about 50% methanol:about 50% acetone combination; (e) about 10% glacialacetic acid:about 30% chloroform:about 60% absolute ethanol combination;and (f) about 50% methanol:about 50% ethanol combination.
 5. The methodof claim 1, wherein the temperature ranges from about −100° C. to about100° C.
 6. The method of claim 1, wherein the mass spectrometry matrixmaterial is α-cyano-4-hydroxycinnamic acid or sinapinic acid or acombination thereof.
 7. The method of claim 1, wherein the tissue sampleis animal tissue.
 8. The method of claim 1, wherein the tissue sample ismammalian tissue.
 9. The method of claim 1, wherein the tissue sample ishuman tissue.
 10. The method of claim 1, wherein the fixative comprisesformaldehyde, glutaraldehyde, acrolein, glyoxal, or malonaldehyde, or acombination thereof.
 11. The method of claim 1, wherein the fixativecomprises methanol or ethanol, or a combination thereof.
 12. The methodof claim 1, wherein the fixative comprises osmium tetroxide or chromicacid.
 13. The method of claim 1, wherein the fixative comprises mercuricchloride.
 14. The method of claim 1, wherein the fixative is selectedfrom the group consisting of Carnoy's fixatives, methacran, Wolman'ssolution, Rossman's fluid, Gendre's fluid, Bouin's fluid, Zenker'sfluid, Helly's fluid, B5 fixative, Susa fluid, Elftman's fixative, Swankand Davenport's fixative, Lillie's alcoholic lead nitrate, andcetylpyridinium chloride.